Tetrazole derivatives of bile acids as FXR/TGR5 agonists and methods of use thereof

ABSTRACT

The present invention provides compounds of Formula I:pharmaceutical compositions comprising these compounds and methods of using these compounds to treat or prevent a disease or disorder mediated by FXR and/or TGR5.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/084,769, filed on Nov. 26, 2014, and U.S. Provisional Application No.62/116,042, filed on Feb. 13, 2015. The entire teachings of the aboveapplications are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates generally to compounds and pharmaceuticalcompositions useful as FXR/TGR5 modulators. Specifically, the presentinvention relates to bile acid derivatives and methods for theirpreparation and use.

BACKGROUND OF THE INVENTION

Farnesoid X Receptor (FXR) is an orphan nuclear receptor initiallyidentified from a rat liver cDNA library (B M. Forman, et al., Cell,1995, 81(5), 687-693) that is most closely related to the insectecdysone receptor. FXR is a member of the nuclear receptor family ofligand-activated transcription factors that includes receptors for thesteroid, retinoid, and thyroid hormones (D J. Mangelsdorf, et al., Cell,1995, 83(6), 841-850). The relevant physiological ligands of FXR arebile acids (D. Parks et al., Science, 1999, 284(5418), 1362-1365). Themost potent one is chenodeoxycholic acid (CDCA), which regulates theexpression of several genes that participate in bile acid homeostasis.Farnesol and derivatives, together called farnesoids, are originallydescribed to activate the rat orthologue at high concentration but theydo not activate the human or mouse receptor. FXR is expressed in theliver, throughout the entire gastrointestinal tract including theesophagus, stomach, duodenum, small intestine, colon, ovary, adrenalgland and kidney. Beyond controlling intracellular gene expression, FXRseems to be also involved in paracrine and endocrine signaling byupregulating the expression of the cytokine Fibroblast Growth Factor (J.Holt et al., Genes Dev., 2003, 17(13), 1581-1591; T. Inagaki et al.,Cell Metab., 2005, 2(4), 217-225).

Small molecule compounds which act as FXR modulators have been disclosedin the following publications: WO 2000/037077, WO 2003/015771, WO2004/048349, WO 2007/076260, WO 2007/092751, WO 2007/140174, WO2007/140183, WO 2008/051942, WO 2008/157270, WO 2009/005998, WO2009/012125, WO 2008/025539, WO 2008/025540, WO 2011/020615, and WO2013/007387.

Further small molecule FXR modulators have been recently reviewed (R. C.Buijsman et al. Curr. Med. Chem. 2005, 12, 1017-1075).

TGR5 receptor is a G-protein-coupled receptor that has been identifiedas a cell-surface receptor that is responsive to bile acids (BAs). Theprimary structure of TGR5 and its responsiveness to bile acids has beenfound to be highly conserved in TGR5 among human, bovine, rabbit, rat,and mouse, and thus suggests that TGR5 has important physiologicalfunctions. TGR5 has been found to be widely distributed in not onlylymphoid tissues but also in other tissues. High levels of TGR5 mRNAhave been detected in placenta, spleen, and monocytes/macrophages. Bileacids have been shown to induce internalization of the TGR5 fusionprotein from the cell membrane to the cytoplasm (Kawamata et al., J.Bio. Chem., 2003, 278, 9435). TGR5 has been found to be identical tohGPCR19 reported by Takeda et al., FEBS Lett. 2002, 520, 97-101.

TGR5 is associated with the intracellular accumulation of cAMP, which iswidely expressed in diverse cell types. While the activation of thismembrane receptor in macrophages decreases pro-inflammatory cytokineproduction, (Kawamata, Y., et al., J. Biol. Chem. 2003, 278, 9435-9440)the stimulation of TGR5 by BAs in adipocytes and myocytes enhancesenergy expenditure (Watanabe, M., et al. Nature. 2006, 439, 484-489).This latter effect involves the cAMP-dependent induction of type 2iodothyronine deiodinase (D2), which by, locally converting T4 into T3,gives rise to increased thyroid hormone activity. Consistent with therole of TGR5 in the control of energy metabolism, female TGR5 knock-outmice show a significant fat accumulation with body weight gain whenchallenged with a high fat diet, indicating that the lack of TGR5decreases energy expenditure and elicits obesity (Maruyama, T., et al.,J. Endocrinol. 2006, 191, 197-205). In addition and in line with theinvolvement of TGR5 in energy homeostasis, bile acid activation of themembrane receptor has also been reported to promote the production ofglucagon-like peptide 1 (GLP-1) in murine enteroendocrine cell lines(Katsuma, S., Biochem. Biophys. Res. Commun., 2005, 329, 386-390). Onthe basis of all the above observations, TGR5 is an attractive targetfor the treatment of disease e.g., obesity, diabetes and metabolicsyndrome.

In addition to the use of TGR5 agonists for the treatment and preventionof metabolic diseases, compounds that modulate TGR5 modulators are alsouseful for the treatment of other diseases e.g., central nervousdiseases as well as inflammatory diseases (WO 01/77325 and WO 02/84286).Modulators of TGR5 also provide methods of regulating bile acid andcholesterol homeostasis, fatty acid absorption, and protein andcarbohydrate digestion.

There is a need for the development of FXR and/or TGR5 modulators forthe treatment and prevention of disease. The present invention hasidentified compounds, which contain an amino, urea, sulfonylurea orsulfonamide moieties, which modulate FXR and/or TGR as well as methodsof using these compounds to treat disease.

SUMMARY OF THE INVENTION

In one aspect, the invention provides compounds represented by FormulaI, or pharmaceutically acceptable salts, stereoisomers, solvates,hydrates or combination thereof:

wherein:X is absent, —C(O)NH— or —NH—R₁ is selected from the group consisting of:

-   -   1) Hydrogen;    -   2) Substituted or unsubstituted —C₁-C₈ alkyl;    -   3) Substituted or unsubstituted —C₂-C₈ alkenyl;    -   4) Substituted or unsubstituted —C₂-C₈ alkynyl;    -   5) Substituted or unsubstituted arylalkyl;    -   6) Substituted or unsubstituted aryl; or        R₁ is selected from the group consisting of:    -   1) Hydrogen;    -   2) Substituted or unsubstituted —C₁-C₈ alkyl;    -   3) Substituted or unsubstituted —C₂-C₈ alkenyl;    -   4) Substituted or unsubstituted —C₂-C₈ alkynyl;    -   5) Substituted or unsubstituted alkylaryl;    -   6) Substituted or unsubstituted aryl;        preferably R₁ is hydrogen or methyl;        m is 0, 1, 2 or 3; preferably m is 0, 1 or 2.        R₂ is hydrogen, hydroxyl, —OSO₃H, —OSO₃ ⁻, —OAc, —OPO₃H₂ or        —OPO₃ ²⁻; preferably R₂ is hydrogen or hydroxyl, more preferably        hydrogen.        R₃ is hydrogen, halogen, CN, N₃, hydroxyl, —OSO₃H, —OSO₃ ⁻,        —OAc, —OPO₃H₂, —OPO₃ ²⁻, —SR₁ or —NHR₁, wherein R₁ is as defined        previously; preferably R₃ is hydrogen.        Or R₂ and R₃ are taken together with the carbon atoms to which        they are attached to form —CH═CH— or cycloalkyl ring or        heterocycloalkyl ring such as, but not limited to cyclopropyl or        epoxide.        R₄ and R₅ are independently selected from hydrogen or hydroxyl        protecting group such as, but not limited to acetyl,        trimethylsilyl, or benzyl; preferably R₄ and R₅ are hydrogen.        R₆ is selected from the group consisting of:    -   1) Hydrogen;    -   2) Halogen;    -   3) Substituted or unsubstituted —C₁-C₈ alkyl;    -   4) Substituted or unsubstituted —C₂-C₈ alkenyl;    -   5) Substituted or unsubstituted —C₂-C₈ alkynyl; and    -   6) Substituted or unsubstituted —C₃-C₈ cycloalkyl;

Preferably R₆ is C₁-C₄-alkyl, more preferably R₆ is ethyl.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundor combination of compounds of the present invention, or apharmaceutically acceptable salt form, stereoisomer, solvate, hydrate orcombination thereof, in combination with a pharmaceutically acceptablecarrier or excipient.

In another embodiment, the present invention provides a method for theprevention or treatment of an FXR mediated disease or condition. Themethod comprises administering a therapeutically effective amount of acompound of formula (I). The present invention also provides the use ofa compound of formula (I) for the preparation of a medicament for theprevention or treatment of an FXR mediated disease or condition.

In yet another embodiment, the present invention provides a method forthe prevention or treatment of a TGR5 mediated disease or condition. Themethod comprises administering a therapeutically effective amount of acompound of formula (I). The present invention also provides the use ofa compound of formula (I) for the preparation of a medicament for theprevention or treatment of a TGR5 mediated disease or condition.

In certain embodiments, a disease that involves modulation of the TGR5receptor is selected from metabolic disease, inflammatory disease, liverdisease, autoimmune disease, cardiac disease, kidney disease, cancer,and gastrointestinal disease.

DETAILED DESCRIPTION OF THE INVENTION

A first embodiment of the invention is a compound represented by FormulaI as described above, or a pharmaceutically acceptable salt, hydrate,solvate, ester or prodrug thereof. In one embodiment of the compounds ofFormula I, R₁ is hydrogen or methyl, R₂ is hydrogen or hydroxyl, R₃, R₄,and R₅ are each hydrogen and R₆ is ethyl. In another embodiment of thecompounds of Formula I, R₁, R₂, R₃, R₄, and R₅ are each hydrogen and R₆is ethyl. In certain embodiments of the compounds of Formula I, R₂ ishydrogen or methyl; m is 0, 1 or 2; R₃ is hydrogen or hydroxyl; R₄ ishydrogen; R₅ is hydrogen; R₆ is hydrogen; R₇ is ethyl; R₈ is hydrogenand R₉ is hydrogen.

In a preferred embodiment, the compounds of the invention have thestereochemistry set forth in Formula IA:

wherein R₁, R₂, R₃, R₄, R₅, R₆, X and m are as defined above.

A second embodiment of the invention is a compound represented byFormula II or a pharmaceutically acceptable salt, hydrate, solvate,ester or prodrug thereof:

wherein R₁, R₂, R₃, R₆, X and m are as previously defined.

A third embodiment of the invention is a compound represented by FormulaIII or a pharmaceutically acceptable salt, hydrate, solvate, ester orprodrug thereof:

wherein R₁, R₂, R₆, X and m are as previously defined.

Illustrative structures of formula (III) can be represented, but notlimited, by formula (III-1˜III-27), where R₆ and m are as previouslydefined; preferably, R₆ is ethyl and m is 0, 1 or 2.

A fourth embodiment of the invention is a compound represented byFormula IV-A or IV-B or a pharmaceutically acceptable salt, solvate,hydrate, ester or prodrug thereof:

wherein m is as previously defined. Preferably, m is 0, 1 or 2.

A fifth embodiment of the invention is a compound represented by FormulaV-A or V-B or a pharmaceutically acceptable salt, solvate, hydrate,ester or prodrug thereof:

wherein m is as previously defined. Preferably, m is 0, 1 or 2.

A sixth embodiment of the invention is a compound represented by FormulaVI-A or VI-B or a pharmaceutically acceptable salt, solvate, hydrate,ester or prodrug thereof:

wherein m is as previously defined. Preferably, m is 0, 1 or 2.

A seventh embodiment of the invention is a compound represented byFormula VII-A or VII-B or a pharmaceutically acceptable salt, solvate,hydrate, ester or prodrug thereof:

wherein m is as previously defined. Preferably, m is 0, 1 or 2.

An eighth embodiment of the invention is a compound represented byFormula VIII-A or VIII-B or a pharmaceutically acceptable salt, solvate,hydrate, ester or prodrug thereof:

wherein m is as previously defined. Preferably, m is 0, 1 or 2.

A ninth embodiment of the invention is a compound represented by FormulaIX-A or IX-B or a pharmaceutically acceptable salt, solvate, hydrate,ester or prodrug thereof:

wherein m is as previously defined. Preferably, m is 0, 1 or 2.

In certain embodiments, the present invention provides a method for theprevention or treatment of an FXR mediated disease or condition. Themethod comprises administering a therapeutically effective amount of acompound of formula (I). The present invention also provides the use ofa compound of formula (I) for the preparation of a medicament for theprevention or treatment of an FXR mediated disease or condition.

In certain embodiments, the FXR-mediated disease or condition iscardiovascular disease, atherosclerosis, arteriosclerosis,hypercholesteremia, or hyperlipidemia chronic liver disease,gastrointestinal disease, renal disease, metabolic disease, cancer(i.e., colorectal cancer), or neurological indications such as stroke.

In certain embodiments, the chronic liver disease is primary biliarycirrhosis (PBC), cerebrotendinous xanthomatosis (CTX), primarysclerosing cholangitis (PSC), drug induced cholestasis, intrahepaticcholestasis of pregnancy, parenteral nutrition associated cholestasis(PNAC), bacterial overgrowth or sepsis associated cholestasis,autoimmune hepatitis, chronic viral hepatitis, alcoholic liver disease,nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis(NASH), liver transplant associated graft versus host disease, livingdonor transplant liver regeneration, congenital hepatic fibrosis,choledocholithiasis, granulomatous liver disease, intra- or extrahepaticmalignancy, Sjogren's syndrome, Sarcoidosis, Wilson's disease, Gaucher'sdisease, hemochromatosis, or alpha 1-antitrypsin deficiency. In certainembodiments, the gastrointestinal disease is inflammatory bowel disease(IBD) (including Crohn's disease and ulcerative colitis), irritablebowel syndrome (IBS), bacterial overgrowth, malabsorption,post-radiation colitis, or microscopic colitis.

In certain embodiments, the renal disease is diabetic nephropathy, focalsegmental glomerulosclerosis (FSGS), hypertensive nephrosclerosis,chronic glomerulonephritis, chronic transplant glomerulopathy, chronicinterstitial nephritis, or polycystic kidney disease.

In certain embodiments, the cardiovascular disease is atherosclerosis,arteriosclerosis, dyslipidemia, hypercholesterolemia, orhypertriglyceridemia.

In certain embodiments, the metabolic disease is insulin resistance,Type I and Type II diabetes, or obesity.

In yet another embodiment, the invention provides the use of thecompound or pharmaceutical composition of the invention, in themanufacture of a medicament for a treating or preventing a disease in asubject that involves modulation of the TGR5 receptor. The inventionincludes a method of treating or preventing a disease that involvesmodulation of the TGR5 receptor in a subject by administering a compoundor pharmaceutical composition of the invention.

In certain embodiments, a disease that involves modulation of the TGR5receptor is selected from metabolic disease, inflammatory disease, liverdisease, autoimmune disease, cardiac disease, kidney disease, cancer,and gastrointestinal disease.

In one aspect, the invention provides for the use, wherein the diseaseis an inflammatory disease selected from allergy, osteoarthritis,appendicitis, bronchial asthma, pancreatitis, allergic rash, andpsoriasis. The invention includes a method of treating or preventing aninflammatory disease selected from allergy, osteoarthritis,appendicitis, bronchial asthma, pancreatitis, allergic rash, andpsoriasis.

In one aspect, the invention provides for the use, wherein the diseaseis an autoimmune disease selected from rheumatoid arthritis, multiplesclerosis, and type I diabetes. The invention includes a method oftreating or preventing an autoimmune disease selected from rheumatoidarthritis, multiple sclerosis, and type I diabetes.

In one aspect, the invention provides for the use, wherein the diseaseis a gastrointestinal disease selected from inflammatory bowel disease(Crohn's disease, ulcerative colitis), short bowel syndrome(post-radiation colitis), microscopic colitis, irritable bowel syndrome(malabsorption), and bacterial overgrowth. The invention includes amethod of treating or preventing a gastrointestinal disease selectedfrom inflammatory bowel disease (Crohn's disease, ulcerative colitis),short bowel syndrome (post-radiation colitis), microscopic colitis,irritable bowel syndrome (malabsorption), and bacterial overgrowth.

In one aspect, the invention provides for the use, wherein the diseaseis kidney disease selected from diabetic nephropathy, chronic renalfailure, hypertensive nephrosclerosis, chronic glomerulonephritis,chronic transplant glomerulopathy, chronic interstitial nephritis, andpolycystic kidney disease. The invention includes a method of treatingor preventing kidney disease selected from diabetic nephropathy, chronicrenal failure, hypertensive nephrosclerosis, chronic glomerulonephritis,chronic transplant glomerulopathy, chronic interstitial nephritis, andpolycystic kidney disease.

In one aspect, the invention provides for the use, wherein the diseaseis cancer selected from colorectal cancer, liver cancer, hepatocellularcarcinoma, cholangio carcinoma, renal cancer, gastric cancer, pancreaticcancer, prostate cancer, and insulanoma. The invention includes a methodof treating or preventing cancer selected from colorectal cancer, livercancer, hepatocellular carcinoma, cholangio carcinoma, renal cancer,gastric cancer, pancreatic cancer, prostate cancer, and insulanoma.

In one aspect, the compound is a selective FXR agonist over TGR5activator.

In one aspect, the compound is a selective TGR5 agonist over FXRactivator.

In one aspect, the compound is a dual agonist for both FXR and TGR5.

Yet a further aspect of the present invention is a process of making anyof the compounds delineated herein employing any of the synthetic meansdelineated herein.

DEFINITIONS

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The term “alkyl”, as used herein, refers to a saturated, monovalentstraight- or branched-chain hydrocarbon group. Preferred alkyl radicalsinclude C₁-C₆ alkyl and C₁-C₈ alkyl radicals. Examples of C₁-C₆ alkylgroups include, but are not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl groups; and examplesof C₁-C₈ alkyl groups include, but are not limited to, methyl, ethyl,propyl, isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, andoctyl groups.

The term “alkenyl”, as used herein, denote a monovalent group derivedfrom a hydrocarbon moiety by the removal of a single hydrogen atomwherein the hydrocarbon moiety has at least one carbon-carbon doublebond. Preferred alkenyl groups include C₂-C₆ alkenyl and C₂-C₈ alkenylgroups. Alkenyl groups include, but are not limited to, for example,ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl andthe like.

The term “alkynyl”, as used herein, denotes a monovalent group derivedfrom a hydrocarbon moiety by the removal of a single hydrogen atomwherein the hydrocarbon moiety has at least one carbon-carbon triplebond. Preferred alkynyl groups include C₂-C₆ alkynyl and C₂-C₈ alkynylgroups. Representative alkynyl groups include, but are not limited to,for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl and thelike.

The term “carbocycle” refers to a saturated (e.g., “cycloalkyl”),partially saturated (e.g., “cycloalkenyl” or “cycloalkynyl”) orcompletely unsaturated (e.g., “aryl”) ring system containing zeroheteroatom ring atom. “Ring atoms” or “ring members” are the atoms boundtogether to form the ring or rings. Where a carbocycle group is adivalent moiety linking two other elements in a depicted chemicalstructure (such as Z in Formula I_(A)), the carbocycle group can beattached to the two other elements through any two substitutable ringatoms. A C₄-C₆ carbocycle has 4-6 ring atoms.

The term “cycloalkyl”, as used herein, denotes a monovalent groupderived from a monocyclic or polycyclic saturated carbocyclic ringcompound by the removal of a single hydrogen atom. Preferred cycloalkylgroups include C₃-C₈ cycloalkyl and C₃-C₁₂ cycloalkyl groups. Examplesof C₃-C₈-cycloalkyl include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; andexamples of C₃-C₁₂-cycloalkyl include, but not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, andbicyclo[2.2.2]octyl.

The term “cycloalkenyl” as used herein, denote a monovalent groupderived from a monocyclic or polycyclic carbocyclic ring compound havingat least one carbon-carbon double bond by the removal of a singlehydrogen atom. Preferred cycloalkenyl groups include C₃-C₈ cycloalkenyland C₃-C₁₂ cycloalkenyl groups. Examples of C₃-C₈-cycloalkenyl include,but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like; and examples ofC₃-C₁₂-cycloalkenyl include, but not limited to, cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,and the like.

The term “aryl,” as used herein, refers to a mono- or bicycliccarbocyclic ring system having one or two aromatic rings including, butnot limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyland the like.

The term “arylalkyl,” as used herein, refers to a C₁-C₃ alkyl or C₁-C₆alkyl residue attached to an aryl ring. Examples include, but are notlimited to, benzyl, phenethyl and the like.

The term “heteroaryl,” as used herein, refers to a mono-, bi-, ortri-cyclic aromatic radical or ring having from five to ten ring atomsof which at least one ring atom is selected from S, O and N; wherein anyN or S contained within the ring may be optionally oxidized. Preferredheteroaryl groups are monocyclic or bicyclic. Heteroaryl groups include,but are not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.

The term “heteroarylalkyl,” as used herein, refers to a C₁-C₃ alkyl orC₁-C₆ alkyl residue attached to a heteroaryl ring. Examples include, butare not limited to, pyridinylmethyl, pyrimidinylethyl and the like.

The term “substituted” as used herein, refers to independent replacementof one, two, or three or more of the hydrogen atoms thereon withsubstituents including, but not limited to, deuterium, —F, —Cl, —Br, —I,—OH, protected hydroxy, —NO₂, —CN, —NH₂, N₃, protected amino, alkoxy,thioalkoxy, oxo, -halo-C₁-C₁₂-alkyl, -halo-C₂-C₁₂-alkenyl,-halo-C₂-C₁₂-alkynyl, -halo-C₃-C₁₂-cycloalkyl, —NH—C₁-C₁₂-alkyl,—NH—C₂-C₁₂-alkenyl, —NH—C₂-C₁₂-alkynyl, —NH—C₃-C₁₂-cycloalkyl, —NH-aryl,—NH-heteroaryl, —NH-heterocycloalkyl, -dialkylamino, -diarylamino,-diheteroarylamino, —O—C₁-C₁₂-alkyl, —O—C₂-C₁₂-alkenyl,—O—C₂-C₁₂-alkynyl, —O—C₃-C₁₂-cycloalkyl, —O-aryl, —O-heteroaryl, —O—heterocycloalkyl, —C(O)—C₁-C₁₂-alkyl, —C(O)—C₂-C₁₂-alkenyl,—C(O)—C₂-C₁₂-alkynyl, —C(O)—C₃-C₁₂-cycloalkyl, —C(O)-aryl,—C(O)-heteroaryl, —C(O)-heterocycloalkyl, —CONH₂, —CONH—C₁-C₁₂-alkyl,—CONH—C₂-C₁₂-alkenyl, —CONH—C₂-C₁₂-alkynyl, —CONH—C₃-C₁₂-cycloalkyl,—CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl,—OCO₂—C₁-C₁₂-alkyl, —OCO₂—C₂-C₁₂-alkenyl, —OCO₂—C₂-C₁₂-alkynyl,—OCO₂—C₃-C₁₂-cycloalkyl, —OCO₂-aryl, —OCO₂-heteroaryl,—OCO₂-heterocycloalkyl, —OCONH₂, —OCONH—C₁-C₁₂-alkyl,—OCONH—C₂-C₁₂-alkenyl, —OCONH—C₂-C₁₂-alkynyl, —OCONH—C₃-C₁₂-cycloalkyl,—OCONH-aryl, —OCONH-heteroaryl, —OCONH-heterocycloalkyl,—NHC(O)—C₁-C₁₂-alkyl, —NHC(O)—C₂-C₁₂-alkenyl, —NHC(O)—C₂-C₁₂-alkynyl,—NHC(O)—C₃-C₁₂-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl,—NHC(O)-heterocycloalkyl, —NHCO₂—C₁-C₁₂-alkyl, —NHCO₂—C₂-C₁₂-alkenyl,—NHCO₂—C₂-C₁₂-alkynyl, —NHCO₂—C₃-C₁₂-cycloalkyl, —NHCO₂-aryl,—NHCO₂-heteroaryl, —NHCO₂-heterocycloalkyl, —NHC(O)NH₂,—NHC(O)NH—C₁-C₁₂-alkyl, —NHC(O)NH—C₂-C₁₂-alkenyl,—NHC(O)NH—C₂-C₁₂-alkynyl, —NHC(O)NH—C₃-C₁₂-cycloalkyl, —NHC(O)NH-aryl,—NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH₂,—NHC(S)NH—C₁-C₁₂-alkyl, —NHC(S)NH—C₂-C₁₂-alkenyl,—NHC(S)NH—C₂-C₁₂-alkynyl, —NHC(S)NH—C₃-C₁₂-cycloalkyl, —NHC(S)NH-aryl,—NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH₂,—NHC(NH)NH—C₁-C₁₂-alkyl, —NHC(NH)NH—C₂-C₁₂-alkenyl,—NHC(NH)NH—C₂-C₁₂-alkynyl, —NHC(NH)NH—C₃-C₁₂-cycloalkyl,—NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl,—NHC(NH)—C₁-C₁₂-alkyl, —NHC(NH)—C₂-C₁₂-alkenyl, —NHC(NH)—C₂-C₁₂-alkynyl,—NHC(NH)—C₃-C₁₂-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl,—NHC(NH)-heterocycloalkyl, —C(NH)NH—C₁-C₁₂-alkyl,—C(NH)NH—C₂-C₁₂-alkenyl, —C(NH)NH—C₂-C₁₂-alkynyl,—C(NH)NH—C₃-C₁₂-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl,—C(NH)NH-heterocycloalkyl, —S(O)—C₁-C₁₂-alkyl, —S(O)—C₂-C₁₂-alkenyl,—S(O)—C₂-C₁₂-alkynyl, —S(O)—C₃-C₁₂-cycloalkyl, —S(O)-aryl,—S(O)-heteroaryl, —S(O)-heterocycloalkyl-SO₂NH₂, —SO₂NH—C₁-C₁₂-alkyl,—SO₂NH—C₂-C₁₂-alkenyl, —SO₂NH—C₂-C₁₂-alkynyl, —SO₂NH—C₃-C₁₂-cycloalkyl,—SO₂NH-aryl, —SO₂NH-heteroaryl, —SO₂NH-heterocycloalkyl,—NHSO₂—C₁-C₁₂-alkyl, —NHSO₂—C₂-C₁₂-alkenyl, —NHSO₂—C₂-C₁₂-alkynyl,—NHSO₂—C₃-C₁₂-cycloalkyl, —NHSO₂-aryl, —NHSO₂-heteroaryl,—NHSO₂-heterocycloalkyl, —CH₂NH₂, —CH₂SO₂CH₃, -aryl, -arylalkyl,-heteroaryl, -heteroarylalkyl, -heterocycloalkyl, —C₃-C₁₂-cycloalkyl,polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH,—S—C₁-C₁₂-alkyl, —S—C₂-C₁₂-alkenyl, —S—C₂-C₁₂-alkynyl,—S—C₃-C₁₂-cycloalkyl, —S-aryl, —S-heteroaryl, —S— heterocycloalkyl,methylthiomethyl, or -L′-R′, wherein L′ is C₁-C₆alkylene,C₂-C₆alkenylene or C₂-C₆alkynylene, and R′ is aryl, heteroaryl,heterocyclic, C₃-C₁₂cycloalkyl or C₃-C₁₂cycloalkenyl. It is understoodthat the aryls, heteroaryls, alkyls, and the like can be furthersubstituted. In some cases, each substituent in a substituted moiety isadditionally optionally substituted with one or more groups, each groupbeing independently selected from —F, —Cl, —Br, —I, —OH, —NO₂, —CN, or—NH₂.

In accordance with the invention, any of the aryls, substituted aryls,heteroaryls and substituted heteroaryls described herein, can be anyaromatic group. Aromatic groups can be substituted or unsubstituted.

It is understood that any alkyl, alkenyl, alkynyl, cycloalkyl andcycloalkenyl moiety described herein can also be an aliphatic group, analicyclic group or a heterocyclic group. An “aliphatic group” isnon-aromatic moiety that may contain any combination of carbon atoms,hydrogen atoms, halogen atoms, oxygen, nitrogen or other atoms, andoptionally contain one or more units of unsaturation, e.g., doubleand/or triple bonds. An aliphatic group may be straight chained,branched or cyclic and preferably contains between about 1 and about 24carbon atoms, more typically between about 1 and about 12 carbon atoms.In addition to aliphatic hydrocarbon groups, aliphatic groups include,for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines,and polyimines, for example. Such aliphatic groups may be furthersubstituted. It is understood that aliphatic groups may be used in placeof the alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynylenegroups described herein.

The term “alicyclic,” as used herein, denotes a monovalent group derivedfrom a monocyclic or polycyclic saturated carbocyclic ring compound bythe removal of a single hydrogen atom. Examples include, but are notlimited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl. Such alicyclic groups maybe further substituted.

The term “heterocycloalkyl” and “heterocyclic” can be usedinterchangeably and refer to a non-aromatic 3-, 4-, 5-, 6- or 7-memberedring or a bi- or tri-cyclic group fused system, where: (i) each ringcontains between one and three heteroatoms independently selected fromoxygen, sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 doublebonds and each 6-membered ring has 0 to 2 double bonds, (iii) thenitrogen and sulfur heteroatoms may optionally be oxidized, (iv) thenitrogen heteroatom may optionally be quaternized, (v) any of the aboverings may be fused to a benzene ring, and (vi) the remaining ring atomsare carbon atoms which may be optionally oxo-substituted. Representativeheterocycloalkyl groups include, but are not limited to, [1,3]dioxolane,pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, andtetrahydrofuryl. Such heterocyclic groups may be further substituted togive substituted heterocyclic.

It will be apparent that in various embodiments of the invention, thesubstituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, arylalkyl, heteroarylalkyl, andheterocycloalkyl are intended to be monovalent or divalent. Thus,alkylene, alkenylene, and alkynylene, cycloalkylene, cycloalkenylene,cycloalkenylene, arylalkylene, hetoerarylalkylene andheterocycloalkylene groups are to be included in the above definitions,and are applicable to provide the formulas herein with proper valency.

The term “hydroxy activating group”, as used herein, refers to a labilechemical moiety which is known in the art to activate a hydroxy group sothat it will depart during synthetic procedures such as in asubstitution or elimination reactions. Examples of hydroxy activatinggroup include, but not limited to, mesylate, tosylate, triflate,p-nitrobenzoate, phosphonate and the like.

The term “activated hydroxy”, as used herein, refers to a hydroxy groupactivated with a hydroxy activating group, as defined above, includingmesylate, tosylate, triflate, p-nitrobenzoate, phosphonate groups, forexample.

The term “protected hydroxy,” as used herein, refers to a hydroxy groupprotected with a hydroxy protecting group, as defined above, includingbenzoyl, acetyl, trimethylsilyl, triethylsilyl, and methoxymethylgroups.

The term “hydroxy protecting group,” as used herein, refers to a labilechemical moiety which is known in the art to protect a hydroxy groupagainst undesired reactions during synthetic procedures. After saidsynthetic procedure(s) the hydroxy protecting group as described hereinmay be selectively removed. Hydroxy protecting groups as known in theare described generally in T. H. Greene and P. G., S. M. Wuts,Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons,New York (1999). Examples of hydroxy protecting groups includebenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl,isopropoxycarbonyl, diphenylmethoxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-(trimethylsilyl)ethoxycarbonyl,2-furfuryloxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl,trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl,2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, 1,1-dimethyl-2-propenyl,3-methyl-3-butenyl, allyl, benzyl, para-methoxybenzyldiphenylmethyl,triphenylmethyl (trityl), tetrahydrofuryl, methoxymethyl,methylthiomethyl, benzyloxymethyl, 2,2,2-trichloroethoxymethyl,2-(trimethylsilyl)ethoxymethyl, methanesulfonyl, para-toluenesulfonyl,trimethylsilyl, triethylsilyl, triisopropylsilyl, and the like.Preferred hydroxy protecting groups for the present invention are acetyl(Ac or —C(O)CH₃), benzoyl (Bz or —C(O)C₆H₅), and trimethylsilyl (TMS or—Si(CH₃)₃).

The terms “halo” and “halogen,” as used herein, refer to an atomselected from fluorine, chlorine, bromine and iodine.

The compounds described herein contain one or more asymmetric centersand thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms that may be defined, in terms of absolutestereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids.The present invention is meant to include all such possible isomers, aswell as their racemic and optically pure forms. Optical isomers may beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques, which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). Whenthe compounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included. Theconfiguration of any carbon-carbon double bond appearing herein isselected for convenience only and is not intended to designate aparticular configuration unless the text so states; thus a carbon-carbondouble bond depicted arbitrarily herein as trans may be cis, trans, or amixture of the two in any proportion.

The term “subject” as used herein refers to a mammal. A subjecttherefore refers to, for example, dogs, cats, horses, cows, pigs, guineapigs, and the like. Preferably the subject is a human. When the subjectis a human, the subject may be referred to herein as a patient.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art.

Berge, et al. describes pharmaceutically acceptable salts in detail inJ. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be preparedin situ during the final isolation and purification of the compounds ofthe invention, or separately by reacting the free base function with asuitable organic acid. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, nontoxic acid addition salts e.g.,salts of an amino group formed with inorganic acids such as hydrochloricacid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloricacid or with organic acids such as acetic acid, maleic acid, tartaricacid, citric acid, succinic acid or malonic acid or by using othermethods used in the art such as ion exchange. Other pharmaceuticallyacceptable salts include, but are not limited to, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

Pharmaceutically acceptable salts can also be prepared by deprotonationof the parent compound with a suitable base, thereby forming the anionicconjugate base of the parent compound. In such salts the counter ion isa cation. Suitable cations include ammonium and metal cations, such asalkali metal cations, including Li⁺, Na⁺, K⁺ and Cs⁺, and alkaline earthmetal cations, such as Mg²⁺ and Ca²⁺.

The term “amino protecting group,” as used herein, refers to a labilechemical moiety which is known in the art to protect an amino groupagainst undesired reactions during synthetic procedures. After saidsynthetic procedure(s) the amino protecting group as described hereinmay be selectively removed. Amino protecting groups as known in the aredescribed generally in T. H. Greene and P. G. M. Wuts, Protective Groupsin Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).Examples of amino protecting groups include, but are not limited to,t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl, and thelike.

As used herein, the term “pharmaceutically acceptable ester” refers toesters of the compounds formed by the process of the present inventionwhich hydrolyze in vivo and include those that break down readily in thehuman body to leave the parent compound or a salt thereof. Suitableester groups include, for example, those derived from pharmaceuticallyacceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic,cycloalkanoic and alkanedioic acids, in which each alkyl or alkenylmoiety advantageously has not more than 6 carbon atoms. Examples ofparticular esters include, but are not limited to, formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

The term “pharmaceutically acceptable prodrugs” as used herein refers tothose prodrugs of the compounds formed by the process of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the present invention. “Prodrug”, as used hereinmeans a compound, which is convertible in vivo by metabolic means (e.g.by hydrolysis) to afford any compound delineated by the formulae of theinstant invention. Various forms of prodrugs are known in the art, forexample, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier(1985); Widder, et al. (ed.), Methods in Enzymology, Vol. 4, AcademicPress (1985); Krogsgaard-Larsen, et al., (ed). “Design and Applicationof Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191(1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988);Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems,American Chemical Society (1975); and Bernard Testa & Joachim Mayer,“Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry AndEnzymology,” John Wiley and Sons, Ltd. (2002).

The term “treating”, as used herein, means relieving, lessening,reducing, eliminating, modulating, or ameliorating, i.e. causingregression of the disease state or condition. Treating can also includeinhibiting, i.e. arresting the development, of a existing disease stateor condition, and relieving or ameliorating, i.e. causing regression ofan existing disease state or condition, for example when the diseasestate or condition may already be present.

The term “preventing”, as used herein means, to completely or almostcompletely stop a disease state or condition, from occurring in apatient or subject, especially when the patient or subject ispredisposed to such or at risk of contracting a disease state orcondition.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar to or comparable in function and appearance tothe reference compound.

The term “aprotic solvent,” as used herein, refers to a solvent that isrelatively inert to proton activity, i.e., not acting as a proton-donor.Examples include, but are not limited to, hydrocarbons, such as hexaneand toluene, for example, halogenated hydrocarbons, such as, forexample, methylene chloride, ethylene chloride, chloroform, and thelike, heterocyclic compounds, such as, for example, tetrahydrofuran andN-methylpyrrolidinone, and ethers such as diethyl ether,bis-methoxymethyl ether. Such solvents are well known to those skilledin the art, and individual solvents or mixtures thereof may be preferredfor specific compounds and reaction conditions, depending upon suchfactors as the solubility of reagents, reactivity of reagents andpreferred temperature ranges, for example. Further discussions ofaprotic solvents may be found in organic chemistry textbooks or inspecialized monographs, for example: Organic Solvents PhysicalProperties and Methods of Purification, 4th ed., edited by John A.Riddick et al., Vol. II, in the Techniques of Chemistry Series, JohnWiley & Sons, NY, 1986.

The terms “protogenic organic solvent” or “protic solvent” as usedherein, refer to a solvent that tends to provide protons, such as analcohol, for example, methanol, ethanol, propanol, isopropanol, butanol,t-butanol, and the like. Such solvents are well known to those skilledin the art, and individual solvents or mixtures thereof may be preferredfor specific compounds and reaction conditions, depending upon suchfactors as the solubility of reagents, reactivity of reagents andpreferred temperature ranges, for example. Further discussions ofprotogenic solvents may be found in organic chemistry textbooks or inspecialized monographs, for example: Organic Solvents PhysicalProperties and Methods of Purification, 4th ed., edited by John A.Riddick et al., Vol. II, in the Techniques of Chemistry Series, JohnWiley & Sons, NY, 1986.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a subject).

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. Additionally, thevarious synthetic steps may be performed in an alternate sequence ororder to give the desired compounds. In addition, the solvents,temperatures, reaction durations, etc. delineated herein are forpurposes of illustration only and variation of the reaction conditionscan produce the desired bridged macrocyclic products of the presentinvention. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein include, for example, those described in R.Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995).

The compounds of this invention may be modified by appending variousfunctionalities via synthetic means delineated herein to enhanceselective biological properties. Such modifications include those whichincrease biological penetration into a given biological system (e.g.,blood, lymphatic system, central nervous system), increase oralavailability, increase solubility to allow administration by injection,alter metabolism and alter rate of excretion.

PHARMACEUTICAL COMPOSITIONS

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the present inventionformulated together with one or more pharmaceutically acceptablecarriers. As used herein, the term “pharmaceutically acceptable carrier”means a non-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Someexamples of materials which can serve as pharmaceutically acceptablecarriers are sugars such as lactose, glucose and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil;safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols;such a propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The pharmaceuticalcompositions of this invention can be administered to humans and otheranimals orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,or drops), buccally, or as an oral or nasal spray.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or: a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

Unless otherwise defined, all technical and scientific terms used hereinare accorded the meaning commonly known to one with ordinary skill inthe art. All publications, patents, published patent applications, andother references mentioned herein are hereby incorporated by referencein their entirety.

Abbreviations

Abbreviations which have been used in the descriptions of the schemesand the examples that follow are:

-   -   ACN for acetonitrile;    -   BME for 2-mercaptoethanol;    -   BOP for benzotriazol-1-yloxy-tris(dimethylamino)phosphonium        hexafluorophosphate;    -   Bu₃SnN₃ for tributyltin azide;    -   BzCl for benzoyl chloride;    -   CDI for carbonyldiimidazole;    -   COD for cyclooctadiene;    -   DABCO for 1,4-diazabicyclo[2.2.2]octane;    -   DAST for diethylaminosulfur trifluoride;    -   DABCYL for        6-(N-4′-carboxy-4-(dimethylamino)azobenzene)-aminohexyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite;    -   DBU for 1, 8-Diazabicycloundec-7-ene;    -   DCC for N,N′-dicyclohexylcarbodiimide;    -   DCM for dichloromethane;    -   DIAD for diisopropyl azodicarboxylate;    -   DIBAL-H for diisobutylaluminum hydride;    -   DIPEA for diisopropyl ethylamine;    -   DMAP for N,N-dimethylaminopyridine;    -   DME for ethylene glycol dimethyl ether;    -   DMEM for Dulbecco's Modified Eagles Media;    -   DMF for N,N-dimethyl formamide;    -   DMSO for dimethylsulfoxide;    -   DSC for N,N′-disuccinimidyl carbonate;    -   DPPA for diphenylphosphoryl azide;    -   DUPHOS for

-   -   EDANS for 5-(2-Amino-ethylamino)-naphthalene-1-sulfonic acid;    -   EDCI or EDC for 1-(3-diethylaminopropyl)-3-ethylcarbodiimide        hydrochloride;    -   Et₂O for diethyl ether;    -   EtOAc for ethyl acetate;    -   EtOH for ethyl alcohol;    -   HATU for O(7-Azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate;    -   HCl for hydrochloric acid;    -   Hoveyda's Cat. for Dichloro(o-isopropoxyphenylmethylene)        (tricyclohexylphosphine)ruthenium(II);    -   In for indium;    -   KHMDS for potassium bis(trimethylsilyl)amide;    -   LAH for lithium aluminium hydride;    -   Ms for mesyl;    -   NaCN for sodium cyanide;    -   NaN₃ for sodiumaizde;    -   NMM for N-4-methylmorpholine;    -   NMI for N-methylimidazole;    -   NMO for N-4-methylmorpholine-N-Oxide;    -   PyBrOP for Bromo-tri-pyrrolidino-phosphonium        hexafluorophosphate;    -   Ph for phenyl;    -   RCM for ring-closing metathesis;    -   RT for reverse transcription;    -   RT-PCR for reverse transcription-polymerase chain reaction;    -   TBME for tert-butyl methyl ether;    -   TEA for triethyl amine;    -   Tf₂O for trifluoromethanesulfonic anhydride;    -   TFA for trifluoroacetic acid;    -   THF for tetrahydrofuran;    -   TLC for thin layer chromatography;    -   (TMS)₂NH for hexamethyldisilazane;    -   TMSOTf for trimethylsilyl trifluoromethanesulfonate;    -   TMSN₃ for trimethylsilyl azide;    -   TBS for t-Butyldimethylsilyl;    -   TMS for trimethylsilyl;    -   TPAP tetrapropylammonium perruthenate;    -   TPP or PPh₃ for triphenylphosphine;    -   TrCl for trityl chloride;    -   DMTrCl for 4,4′-dimethoxytrityl chloride;    -   tBOC or Boc for tert-butyloxy carbonyl.        Synthetic Methods

The compounds and processes of the present invention will be betterunderstood in connection with the following synthetic schemes thatillustrate the methods by which the compounds of the invention may beprepared, which are intended as an illustration only and not to limitthe scope of the invention. Various changes and modifications to thedisclosed embodiments will be apparent to those skilled in the art andsuch changes and modifications including, without limitation, thoserelating to the chemical structures, substituents, derivatives, and/ormethods of the invention may be made without departing from the spiritof the invention and the scope of the appended claims.

As shown in Scheme 1, novel bile acid analogs of the tetrazole compoundof formula (1-6) are prepared from the compound of formula (1-1),wherein m and R₆ are defined as previously. The compound of formula(1-1) is converted to the methyl ester compound of formula (1-2) throughesterification. Then the compound of formula (1-2) is partially reducedto the aldehyde compound of formula (1-3) using suitable reagent suchas, but not limited to, DIBAL-H. The reaction solvent can be, but notlimited to, Et₂O, THF, DCM and toluene. The preferred solvent is Et₂O.The reaction temperature is −78° C. Subsequent reaction of formula (1-3)with hydroxylamine hydrochloride followed by dehydration afford thenitrile compound of formula (1-5), which is then converted to thetetrazole compound of formula (1-6) using suitable reagent such as, butnot limited to, NaN₃, TMSN₃ and Bu₃SnN₃.

An alternative procedure to prepare tetrazole compound of formula (2-4)is illustrated in scheme 2, wherein m and R₆ are defined as previously.The compound of formula (1-1) is converted to the alcohol compound offormula (2-1) using suitable reagent such as, but not limited to, LAH.Subsequent mesylation followed by replacement reaction provide thenitrile compound of formula (2-3), which is then converted to thetetrazole compound of formula (2-4) using suitable reagent such as, butnot limited to, NaN₃, TMSN₃ and Bu₃SnN₃.

Scheme 3 illustrates the preparation of the amino tetrazole compound offormula (3-1) from the compound of formula (2-2), wherein m and R₆ aredefined as previously. The compound of formula (2-2) is converted to thecompound of formula (3-1) by replacement of 5-aminotetrazole.

Another procedure to prepare the tetrazole compound of formula (4-7) isillustrated in scheme 4, wherein m and R₆ are defined as previously, P₁and P₂ are hydroxyl protecting groups. Thus, the two hydroxyl groups ofthe compound of formula (1-1) are protected with P₁ and P₂ groups toafford the compound of formula (4-1). P₁ and P₂ can be same ordifferent. P₁ and P₂ can be any hydroxyl protecting group such as, butnot limited to Ac, Bz, chloroacetyl, TES, TBS, MOM and Bn. The compoundof formula (4-1) is subjected to suitable reduction conditions to givethe compound of formula (4-2), which then is converted to the compoundof formula (4-3) using suitable oxidation conditions followed byolefination to afford compound of formula (4-4). The subsequenthydrogenation provides compound of formula (4-5), which is thenconverted to the tetrazole compound of formula (4-6) using suitablereagent such as, but not limited to, NaN₃, TMSN₃ and Bu₃SnN₃. Furtherdeprotection of the compound of formula of (4-6) gives the tetrazolecompound of formula (4-7). A more detailed discussion of the procedures,reagents and conditions for deprotection of hydroxyl protecting groupsis described in literature, for example, by T. W. Greene and P. G. M.Wuts in “Protective Groups in Organic Synthesis” 3^(rd) ed., John Wiley& Son, Inc., 1999.

Scheme 5 illustrates the preparation of the tetrazole amide compound offormula (5-3) from the compound of formula (1-1), wherein m and R₆ aredefined as previously, P is hydroxyl protecting groups. Thus, theindicated hydroxyl group of the compound of formula (1-1) are protectedwith P group to afford the compound of formula (5-1). P can be anyhydroxyl protecting group such as, but not limited to Ac, Bz,chloroacetyl, TES, TBS, MOM and Bn. A more detailed discussion of theprocedures, reagents and conditions for protection of hydroxyl group isdescribed in literature, for example, by T. W. Greene and P. G. M. Wutsin “Protective Groups in Organic Synthesis” 3^(rd) ed., John Wiley &Son, Inc., 1999. The compound of formula (5-1) is coupled with5-aminotetrazole using suitable coupling condition to provide tetrazoleamide compound of formula (5-2). The coupling reagent can be selectedfrom, but not limited to, DCC, EDC, CDI, di-isopropyl carbodiimide,BOP-Cl, PyBOP, PyAOP, TFFH and HATU. Suitable bases include, but are notlimited to, triethylamine, diisopropylethylamine, DBU,N-methylmorpholine and DMAP. The coupling reaction is carried out in anaprotic solvent such as, but not limited to, CH₂Cl₂, DMF or THF. Thereaction temperature can vary from 0° C. to about 50° C. Thendeprotection of P groups affords the compound of formula (5-3). A moredetailed discussion of the procedures, reagents and conditions fordeprotection of hydroxyl protecting groups is described in literature,for example, by T. W. Greene and P. G. M. Wuts in “Protective Groups inOrganic Synthesis” 3^(rd) ed., John Wiley & Son, Inc., 1999.

EXAMPLES

The compounds and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

Example 1

Step 1-1:

To a 250 mL round bottle flask were added 6(α)-ethyl-chenodeoxycholicacid (6-ECDCA) (960 mg, 2.28 mmol), MeOH (30 mL) and conc. H₂SO₄ (0.1mL) respectively and the reaction mixture was stirred at roomtemperature for 22 h. Transferred to a separation funnel, diluted withEtOAc (150 mL) and washed with aqueous NaHCO₃ (100 mL). The aqueouslayer was extracted with EtOAc (100 mL×2). The combined organic layerswere washed with brine-water (100 mL, 1:1 v/v), dried over Na₂SO₄,filtered and concentrated under reduced pressure. The resulting whitesolid was directly used in the subsequent reaction without any furtherpurification.

Step 1-2:

Methyl ester (1) (848 mg, 1.95 mmol) was dissolved in Et₂O (20 mL) andcooled to −78° C. To the solution was slowly added DIBAL-H (1.0 M inhexane, 5.95 mL, 5.95 mmol). The mixture was allowed to stir at −78° C.for 1 h before carefully diluted with EtOAc (50 mL) and quenched with10% aqueous solution of Rochelle's salt (50 mL) at 0° C. The solutionwas then allowed to warm to room temperature and stir for additional 1hour. Then the aqueous layer was then separated and extracted with EtOAc(100 mL×3). The combined organic layers were washed with brine-water(100 mL, 1:1 v/v), dried over Na₂SO₄, filtered and concentrated underreduced pressure. The resulting pale yellow oil was purified byCombiFlash (40 g SiO₂, EtOAc/hexanes=0→50%) to give the aldehydecompound (2) as a white solid, 755.2 mg, 95% yield.

Step 1-3:

The aldehyde compound (2) (84.7 mg, 0.209 mmol), hydroxylaminehydrochloride (32.0 mg, 0.461 mmol) and NaOAc (39.4 mg, 0.48 mmol) wasdissolved in anhydrous EtOH (2.1 mL). The reaction was allowed to stirfor 1 hour. The solvent was removed under reduced pressure. Water (20mL) was then added and extracted with DCM (40 mL×3). The combinedorganic layers were washed with brine-water (30 mL, 1:1 v/v), dried overNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas directly used in the subsequent reaction without any furtherpurification.

Step 1-4:

The oxime compound (3) obtained from previous step was dissolved inCH₃CN (2.0 mL) followed by addition of Et₃N (116.5 μL, 0.836 mmol) andCDI (135.6 mg, 0.836 mmol) at room temperature. The mixture was allowedto stir at 80° C. for 90 min. The reaction was diluted with water (2 mL)and cooled to 0° C., followed by addition of 1.0 N NaOH (0.2 mL). Thereaction mixture was allowed to slowly warm to room temperature over 90min. CH₃CN was then removed under reduced pressure. The resultingaqueous solution was extracted with EtOAc (20 mL×4). The combinedorganic layers were washed with brine-water (30 mL, 1:1 v/v), dried overNa₂SO₄, filtered and concentrated under reduced pressure. The resultingpale yellow oil was purified by CombiFlash (12 g SiO₂,EtOAc/Hexanes=0→50%) to give the nitrile compound (4) as a white solid,58.6 mg, 70% yield over 2 steps.

Step 1-5:

The nitrile compound (4) (70 mg, 0.174 mmol), NaN₃ (45.3 mg, 0.697 mmol)and triethylamine hydrochloride (71.9 mg 0.522 mmol) was dissolved inDMF (3.0 mL). The reaction was allowed to stir at 160° C. for 30 hours.The reaction was cooled to room temperature and diluted with EtOAc (20mL) and washed with water (20 mL×4). The organic layer was washed withbrine-water (10 mL, 1:1 v/v), dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The resulting pale yellow oil waspurified by CombiFlash (4 g SiO₂, MeOH/DCM=0→5%) to give the tetrazolecompound of example 1 as a white solid, 18.7 mg, 24% yield.

Example 2

Step 2-1:

To a solution of 6(α)-ethyl-chenodeoxycholic acid (6-ECDCA) (5.3 g, 12.6mmol) in THF (160 mL) was slowly added 1.0 M LAH in Et₂O solution (25.2mL, 25.2 mmol) at room temperature. Then the solution was allowed tostir for 6 hours at reflux before carefully quenched with water (1.0 mL)at 0° C. followed by 15% aqueous solution of NaOH (1.0 mL). The solutionwas then allowed to stir for 15 min before addition of another portionof water (2.5 mL). After additional 15 min stirring, the crude was driedover MgSO₄, filtered and washed with EtOAc. The solvent was then removedunder reduced pressure to afford the compound (5) as a white solid,which was directly used in the subsequent reaction without any furtherpurification.

Step 2-2:

The compound (5) obtained from previous step was dissolved in DCM (100mL) followed by addition of Et₃N (2.64 mL, 18.9 mmol) and MsCl (0.9 mL,11.6 mmol) at 0° C. The reaction was allowed to stir for additional 1.5hr at the same temperature. The solvent was removed under reducedpressure. Water (50 mL) was then added and extracted with EtOAc (100mL×3). The combined organic layers were washed with brine-water (50 mL,1:1 v/v), dried over Na₂SO₄, filtered and concentrated under reducedpressure. The residue was directly used in the subsequent reactionwithout any further purification.

Step 2-3:

The compound (6) obtained from previous step was dissolved in DMF (50mL) followed by addition of NaCN (1.14 g, 23.2 mmol) at roomtemperature. The reaction was allowed to stir for 2 days at 60° C. Thereaction was then cooled to temperature, diluted with EtOAc (150 mL) andwashed with water (125 mL×4). The organic layer was washed withbrine-water (30 mL, 1:1 v/v), dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The resulting pale yellow oil waspurified by CombiFlash (120 g SiO₂, EtOAc/hexanes=0→50%) to give thenitrile compound (7) as a white solid, 1.944 g, 54% overall yield.

Step 2-4:

To a pressure released vial was charged compound (7) (128.7 mg, 0.31mmol) and Bu₃SnN₃ (0.339 mL, 1.24 mmol) in toluene (2.1 mL) at roomtemperature. The reaction was allowed to stir for 20 hours at refluxbehind blast shield. The reaction was then cooled to temperature,diluted with EtOAc (20 mL) and washed with water (50 mL). The aqueouslayer was acidified to pH=2 using 1.0 N HCl and then extracted withEtOAc (50 mL×3). The combined organic layers were then washed withbrine-water (50 mL, 1:1 v/v), dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The resulting pale yellow oil waspurified by CombiFlash (12 g SiO₂, MeOH/DCM=0→10%) to give the tetrazolecompound of example 2 as a white solid, 83.7 m g, 59% yield.

Example 3

Step 3-1:

Into a 1000 mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of 6-ECDCA (18.0 g, 0.04mol, 1.00 eq) in THF (200 mL), TEA (86.5 g, 0.86 mol, 20.0 eq),4-dimethylaminopyridine (0.63 g, 0.004 mol, 0.1 eq), and aceticanhydride (87.3 g, 0.86 mol, 20.0 eq). The resulting solution wasstirred at 90° C. for 15 hours. After being cooled to rt, it wasconcentrated and the residue was dissolved in ethyl acetate (500 mL),then was washed with water (100 mL×2), saturated NaCl (100 mL×2). Theorganic layer was dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by flash silica chromatography, elution gradient 0to 20% EtOAc in petroleum ether to give the desired compound (8) as ayellow solid, 20.0 g, 93% yield.

Step 3-2:

Into a solution of compound (8) (20.0 g, 40 mmol) in TFA (150 mL) wasadded TFAA (63.0 g, 300 mmol). Then NaNO₂ was added in 5 portions over45 minutes at 0° C. After stirred at 0° C. for 1 hour, the solution wasmoved to 40° C. for 40 minutes. The solution was quenched with waterafter being cooled to rt, then extracted with ethyl acetate (200 mL×3).The organic layer was dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by flash silica chromatography, elution gradient 10to 30% EtOAc in petroleum ether to give the desired compound (9) as ayellow solid, 12.2 g, 67% yield.

Step 3-3:

Into a 50-mL round-bottom flask, was placed a solution of compound (9)(300 mg, 0.64 mmol), azidotrimethylsilane (3.7 g, 32 mmol),dibutylstannanone (1.5 g, 6.4 mmol) in toluene (15 mL). The resultingsolution was stirred at 120° C. for 15 hours. After cooling to rt thesolution was diluted with 100 mL of water and extracted with ethylacetate (50 mL×3). The combined organic layer was concentrated undervacuum to give the desired compound (10) as a yellow oil, which was usedfor next step without further purification.

Step 3-4:

Into a 50-mL round-bottom flask, was placed a solution of compound (10)(200 mg, 0.42 mmol), methanol (15 mL) and 30% potassium hydroxide (15mL). The resulting solution was stirred at 90° C. for 15 hours. Aftercooling to rt the pH value of the solution was adjusted to 6 with conHCl and extracted with ethyl acetate (20 mL×3). The combined organiclayer was concentrated and purified by Flash-Prep-HPLC ((IntelFlash-1):Column, C18; mobile phase, MeCN/H₂O, Detector, UV 220 nm) to give thetetrazole compound of example 3 as a white solid, 26 mg, 14% yield.

Example 4

Step 4-1:

Ethyl carbonochloridate (1.41 g, 13 mmol) was added dropwise to asolution of compound (8) (5.1 g, 10 mmol) and Et₃N (1.32 g, 13 mmol) indry THF (50 mL) at RT over a period of 10 minutes under nitrogen. Theresulting mixture was stirred at RT for 0.5 hour. The reaction mixturewas cooled to 0° C. and NaBH₄ (1.9 g, 50 mmol) was added slowly. ThenMeOH (15 mL) was added slowly and stirred for 2 hours at 0° C. Theresulting mixture was added water (100 mL), adjusted pH 6 with 2N HCland extracted by EtOAc (200 mL). The organic layer was dried overNa₂SO₄, filtered and evaporated to afford crude product. The crudeproduct was purified by flash silica chromatography, elution gradient 20to 50% EtOAc in petroleum ether. Pure fractions were evaporated todryness to afford compound (11) as a yellow oil, 4.1 g, 68% yield.

Step 4-2:

To a solution of compound (11) (3.9 g, 8 mmol) in CH₂Cl₂ (100 mL),2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) (31 mg, 0.2mol) and KBr (105 mg) were added gradually with ice-bath cooling. To theabove mixture, a solution of NaOCl (8 mL) dissolved in a buffer solution(pH 8.5, 60 mL) prepared from 0.5 M sodium bicarbonate, and 0.05 Msodium carbonate was added. The mixture was stirred at 0° C. for 20 min.The reaction was monitored by TLC using hexane-EtOAc (1:1, v/v) as thedeveloping solvent. After adding methanol (10 mL), the reaction productwas extracted with CH₂Cl₂ (100 mL). The combined organic phase waswashed with water and evaporated to dryness to give the crude compound.The crude product was purified by flash silica chromatography, elutiongradient 20 to 50% EtOAc in petroleum ether. Pure fractions wereevaporated to dryness to afford compound (12) as a yellow oil, 3 g, 77%yield.

Step 4-3:

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed diethyl(cyanomethyl)phosphonate (4.43g, 25.01 mmol) and THF (20 mL). Then sodium hydride (1 g, 41.67 mmol)was added slowly at 0° C. The solution was stirred for 30 minutes. Thena solution of compound (12) (1.22 g, 2.50 mmol) in THF (10 mL) was addedat 0° C. The resulting solution was stirred at 25° C. for 2 hours. Theresulting solution was diluted with 50 mL of water and extracted withEtOAc (50 mL×3). The combined organic layer was washed sequentially withwater (50 mL) and saturated brine (50 mL), then dried over Na₂SO₄,filtered and evaporated. The residue was purified by flash silicachromatography, elution gradient 20 to 50% EtOAc in petroleum ether.Pure fractions were evaporated to dryness to afford the desired compound(13) as yellow oil, 0.7 g, 54% yield.

Step 4-4:

Into a 50-mL round-bottom flask, was placed a solution of compound (13)(52 mg, 0.10 mmol) in methanol (10 mL), then followed by the addition ofPd/C (0.2 g). The resulting solution was stirred under H₂ (1 atm) at rtfor 2 hours. Then Pd/C was removed by filtration and the filtrate wasevaporated to give the desired compound (14) as a yellow oil, which wasused for the next step without further purification.

Step 4-5:

Into a 50-mL round-bottom flask, was placed a solution of compound (14)(300 mg, 0.58 mmol), azidotrimethylsilane (3.4 g, 29.51 mmol), anddibutylstannanone (1.5 g, 6.03 mmol) in toluene (20 mL). The resultingsolution was stirred at 120° C. for 15 hours. After cooling to rt theresulting solution was diluted with 200 mL of water and extracted withethyl acetate (30 mL×3). The organic layer was concentrated under vacuumto give the desired compound (15) as a yellow oil, which was used fornext step without further purification.

Step 4-6:

Into a 50-mL round-bottom flask, was placed a solution of compound (15)(200 mg, 0.39 mmol), methanol (15 mL) and 30% potassium hydroxide (15mL). The resulting solution was stirred at 90° C. for 15 hours. Aftercooling to rt the pH value of the solution was adjusted to 6 with conHCl and extracted with ethyl acetate (20 mL×3). The combined organiclayer was concentrated and purified by Flash-Prep-HPLC ((IntelFlash-1):Column, C18; mobile phase, MeCN/H₂O, Detector, UV 220 nm) to give thetetrazole compound of example 4 as a white solid, 27 mg, 15% yield.

Example 5

Step 5-1:

To a solution of the (methoxymethyl)triphenylphosphonium chloride (3.5g, 10 mmol) in THF (20 mL), NaNHMDS (1M in THF, 10 mL) was added withice-bath cooling. After the mixture was stirred at 0° C. for 20 min, asolution of compound (12) (976 mg, 2 mmol) in THF (5 mL) was addedslowly at 0° C. and stirred for 2 hours at rt. After adding water (100mL), the reaction product was extracted with EtOAc (100 mL). The organiclayer was washed with water (50 mL) and evaporated to dryness to givethe crude compound. The crude product was purified by flash silicachromatography, elution gradient 20 to 50% EtOAc in petroleum ether.Pure fractions were evaporated to dryness to afford compound (16) as ayellow oil, 520 mg, 50% yield.

Step 5-2:

A solution of compound (16) (1.03 g, 2 mmol) and 10N HCl (20 mL) in THF(20 mL) was stirred at 50° C. for 2 hours. Then this mixture was cooledto rt, extracted by EtOAc (80 mL). The organic layer was dried by Na₂SO₄and condensed to get the desired compound (17) as a yellow oil, whichwas used in the next step without further purification.

Step 5-3:

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed diethyl(cyanomethyl)phosphonate (2.12g, 12 mmol) and THF (20 mL). Then sodium hydride (0.48 g, 12 mmol) wasadded slowly at 0° C. This solution was stirred for 30 minutes. Then asolution of compound (17) (0.6 g, 1.2 mmol) in THF (10 mL) was added.The resulting solution was stirred for 2 hours at 25° C. After coolingto rt the solution was diluted with 50 mL of water and extracted withethyl acetate (20 mL×3). The combined organic layer was washed withwater (50 mL) and saturated brine (50 mL), then dried over Na₂SO₄,filtered and evaporated. The residue was purified by flash silicachromatography, elution gradient 20 to 50% EtOAc in petroleum ether.Pure fractions were evaporated to dryness to afford the desired compound(18) as a yellow oil, 0.35 g, 55% yield.

Step 5-4:

Into a 50-mL round-bottom flask, was placed a solution of compound (18)(600 mg, 1.2 mmol) in methanol (10 mL). This resulting mixture wasfollowed by the addition of Pd/C (0.3 g). The resulting solution wasstirred under H₂ (1 atm) at 25° C. for 2 hours. Then Pd/C was removed byfiltration and the filtrate was evaporated to give the desired compound(19) as a yellow oil, which was used for the next step without furtherpurification.

Step 5-5:

Into a 50-mL round-bottom flask, was placed a solution of compound (19)(106 mg, 0.2 mmol), azidotrimethylsilane (1.2 g, 10 mmol), anddibutylstannanone (0.5 g, 2 mmol) in toluene (10 mL). The resultingsolution was stirred at 120° C. for 15 hours. After cooling to rt theresulting solution was diluted with 100 mL of water and extracted withethyl acetate (50 mL×3). The combined organic layer was concentratedunder vacuum to give the desired compound (20) as a yellow oil, whichwas used for next step without further purification.

Step 5-6:

Into a 50-mL round-bottom flask, was placed a solution of compound (20)(200 mg, 0.35 mmol), methanol (15 mL) and 30% potassium hydroxide (15mL). The resulting solution was stirred at 90° C. for 15 hours. Aftercooling to rt the pH value of the solution was adjusted to 6 with conc.HCl and extracted with ethyl acetate (20 mL×3). The combined organiclayer was concentrated and purified by Flash-Prep-HPLC ((IntelFlash-1):Column, C18; mobile phase, MeCN/H₂O, Detector, UV 220 nm) to give thetitle tetrazole compound of example 5 as a white solid, 23 mg, 14%yield.

Example 6

Step 6-1:

To a solution of 6-ECDCA (4.21 g, 10.0 mmol) in DCM at 0° C. (100 mL)was added DIPEA (7.75 g, 60 mmol) followed by MEMCl (3.69 mL, 30 mmol).The reaction mixture was allowed to warm up to RT and stirred overnight,quenched with 1N HCl/brine (1/1), and extracted with EtOAc. The combinedorganic layers were washed with brine and concentrated. The resultingcrude oil was used for next step without further purification.

Step 6-2:

To a stirred solution of above obtained crude compound (21) in MeOH/H₂O(v:v=21/1, 22 mL) at RT was added 50% aq. NaOH (2 mL, 24 mmol). Thereaction mixture was heated to 50° C. and stirred for 3 h, then quenchedwith 1N HCl/brine (1/1), and extracted with EtOAc. The combined organiclayers were washed with brine and concentrated. The residue was purifiedby chromatography on silica gel using acetone/hexane (0→40%) to affordcompound (22) as a pale yellow solid, 4.7 g, 93% yield over 2 steps.

Step 6-3:

To a solution of compound (22) (65 mg, 0.128 mmol) in 2 mL DMF/CH₂Cl₂solution (1:1, v/v) was charged 5-Amino-1,2,3,4-tetrazole (32.6 mg,0.383 mmol), EDCI (49 mg, 0.256 mmol) and DMAP (31.2 mg, 0.256 mmol).The resulting solution was allowed to stir at 60° C. until completionindicated by TLC. The solvent was removed under reduced pressure. Water(15 mL) and 1N HCl aq. solution (2 mL) was added. The solution was thenextracted with ethyl acetate (30 mL×3). The combined organic layer wasdried over Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by CombiFlash (12 g SiO₂, MeOH/CH₂Cl₂=0→10%) togive the compound (23) as a white solid, 30.9 mg, 42% yield.

Step 6-4:

The above obtained compound (23) (30.9 mg) was dissolved in THF (0.9 mL)followed by addition of 0.1 mL of 37% conc. aq. HCl. The mixture wasstirred at room temperature for 1 hour, quenched with sat. NaHCO₃, andextracted with EtOAc (3×). The combined organic layers were dried overNa₂SO₄, filtered and concentrated in vacuo. The residue was byCombiFlash (4 g SiO₂, MeOH/CH₂Cl₂=0→20%) to provide the compound ofexample 6 as white solid, 13.3 mg, 51% yield.

TABLE 1 Exam- ple structure HRMS: ¹H NMR (500 MHz) 1

489.2849 (M + formate)⁻ 2

457.2944 (M − H)⁻ 3.72 (1H, s), 3.46 (1H, br s), 3.03 (1H, m), 2.95 (1H,m), 1.87 (5H, m), 1.70 (4H, m), 1.59 (2H, m), 1.39 (9H, m), 1.22 (3H,m), 1.10 (3H, m), 1.01 (1H, t, J = 8.0 Hz), 0.92 (3H, d, J = 7.0 Hz),0.89 (6H, s), 0.63 (3H, s). 3

429.40 (M − H)⁻ 4

471.45 (M − H)⁻ 5

485.50 (M − H)⁻ 6

486.28 (M − H)⁻.AssaysHuman FXR (NR1H4) Assay

Determination of a ligand mediated Gal4 promoter driven transactivationto quantify ligand binding mediated activation of FXR. FXR ReporterAssay kit purchased from Indigo Bioscience (Catalogue number: IB00601)to determine the potency and efficacy of compound developed by Enantathat can induce FXR activation. The principal application of thisreporter assay system is to quantify functional activity of human FXR.The assay utilizes non-human mammalian cells, CHO (Chinese hamsterovary) cells engineered to express human NR1H4 protein (referred to asFXR). Reporter cells also incorporate the cDNA encoding beetleluciferase which catalyzes the substrates and yields photon emission.Luminescence intensity of the reaction is quantified using aplate-reading luminometer, Envision. Reporter Cells include theluciferase reporter gene functionally linked to an FXR responsivepromoter. Thus, quantifying changes in luciferase expression in thetreated reporter cells provides a sensitive surrogate measure of thechanges in FXR activity. EC₅₀ and efficacy (normalize to CDCA set as100%) is determined by XLFit. The assay is according to themanufacturer's instructions. In brief, the assay was performed in white,96 well plates using final volume of 100 ul containing cells withdifferent doses of compounds. Retrieve Reporter Cells from −80° C.storage. Perform a rapid thaw of the frozen cells by transferring a 10ml volume of 37° C. cell recovery medium into the tube of frozen cells.Recap the tube of Reporter Cells and immediately place it in a 37° C.water bath for 5-10 minutes. Retrieve the tube of Reporter CellSuspension from the water bath. Sanitize the outside surface of the tubewith a 70% alcohol swab, and then transfer it into the cell culturehood. Dispense 90 μl of cell suspension into each well of the 96-wellAssay Plate. Transfer the plate into 37° C. incubator, allowing thecells adherent to the bottom of the well. Dilute compounds in DilutionPlate (DP), and administrate to cells at Assay Plate (AP). DMSO contentof the samples was kept at 0.2%. Cells were incubated for additional 22hours before luciferase activities were measured. Thirty minutes beforeintending to quantify FXR activity, remove Detection Substrate andDetection Buffer from the refrigerator and place them in a low-lightarea so that they may equilibrate to room temperature. Remove theplate's lid and discard all media contents by ejecting it into anappropriate waste container. Gently tap the inverted plate onto a cleanabsorbent paper towel to remove residual droplets. Cells will remaintightly adhered to well bottoms. Add 100 μl of luciferase detectionreagent to each well of the assay plate. Allow the assay plate to restat room temperature for at least 5 minutes following the addition ofLDR. Set the instrument (Envision) to perform a single 5 second “plateshake” prior to reading the first assay well. Read time may be 0.5second (500 mSec) per well. EC₅₀ and Efficacy (normalize to CDCA set as100%) is determined by XLFit.

In Vitro Human TGR5 (GPBAR1) Activity Assay

The potency and efficacy of the compounds of the invention on TGR5receptor can be evaluated using in vitro assays which carried out usingthe express kit from DiscoverX (cAMP Hunter™ eXpress GPBAR1 CHO-K1 GPCRAssay; Catalogue number: 95-0049E2CP2S)GPBAR1 (G protein-coupled bileacid receptor 1) encodes a member of the G protein-coupled receptor(GPCR) superfamily. GPBAR1 activation following ligand binding initiatesa series of second messenger cascades that result in a cellularresponse. Treatment of CHO cells expressing GPBAR1 with bile acidsinduces the production of intracellular cAMP and internalization of thereceptor. The potency and efficacy of compound for GPBAR1 activation bymeasuring cyclic adenosine monophosphate (cyclic AMP or cAMP) levels inlive cells using a competitive immunoassay based on Enzyme FragmentComplementation (EFC).

In briefly, following seeding the cells into the white, 96 wellmicroplate, place it in a 37° C., 5% CO2 in a humidified incubator for18-24 hours prior to testing. On second day, proceed to the appropriatecAMP Hunter eXpress Protocol according to the manufacturer'sinstructions. Dissolve agonist compound in DMSO at the desired stockconcentration, and prepare 3-fold serial dilutions of agonist compoundin Cell Assay Buffer. The concentration of each dilution should beprepared at 4× of the final screening concentration (i.e. 15 μLcompound+45 μL Cell Assay Buffer/cAMP Antibody Reagent). For eachdilution, the final concentration of solvent should remain constant.Transfer 15 μL diluted compound the assay plate and incubate the platefor 30 minutes at 37° C. Following agonist incubation, add 60 μL ofworking cAMP detection reagents/cAMP Solution mixture (cAMP LysisBuffer, Substrate Reagent 1, cAMP Solution D) to the appropriate wells.Incubate for 1 hour at room temperature (23° C.), protected from light.Add 60 μl of cAMP Solution A to the appropriate wells. Incubate for 3hours at room temperature (23° C.), protected from light. Read sampleson Envision standard luminescence plate reader. Calculate of averageEC₅₀ after logarithm transformation.

To assess the FXR agonistic potency of the example compounds as well asfor reference compound, potency ranges were determined in the Human FXR(NR1H4) Assay as listed below in Table 2. The efficacy was normalized toCDCA set as 100%. (A=0.1 μM<EC50<1.0 μM; B=1.0 μM<EC50<10 μM; C=EC50>10μM)

TABLE 2 Example EC50 (μM) Efficacy (%) CDCA C 100 6-ECDCA A 430 1 B 3972 A 354 3 B 192 4 A 234 5 A 191 6 B 105 CDCA = chenodoxycholic acid;6-ECDCA = 6-α-ethylchenodoxycholic acid.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A compound represented by Formula I,

or a pharmaceutically acceptable salt or ester thereof, wherein: X isabsent and m is 2 to 5; R₁ is hydrogen; R₂ is hydrogen; R₃ is hydrogen;R₄ and R₅ are independently selected from hydrogen and hydroxylprotecting groups; and R₆ is ethyl.
 2. The compound of claim 1, whereinR₄ and R₅ are each hydrogen.
 3. A method for the treatment of anFXR-mediated disease or condition in a mammal comprising administeringto the mammal suffering from an FXR-mediated disease or condition atherapeutically effective amount of a compound according to claim
 1. 4.The method according to claim 3, wherein the FXR-mediated disease orcondition is selected from the group consisting of chronic liverdisease, gastrointestinal disease, renal disease, cardiovasculardisease, and metabolic disease.
 5. The method according to claim 4,wherein the chronic liver disease is selected from the group consistingof primary biliary cirrhosis (PBC), cerebrotendinous xanthomatosis(CTX), primary sclerosing cholangitis (PSC), drug induced cholestasis,intrahepatic cholestasis of pregnancy, parenteral nutrition associatedcholestasis (PNAC), bacterial overgrowth or sepsis associatedcholestasis, autoimmune hepatitis, chronic viral hepatitis, alcoholicliver disease, nonalcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), liver transplant associated graft versus hostdisease, living donor transplant liver regeneration, congenital hepaticfibrosis, choledocholithiasis, granulomatous liver disease, intra- orextrahepatic malignancy, Sjogren's syndrome, Sarcoidosis, Wilson'sdisease, Gaucher's disease, hemochromatosis, and alpha 1-antitrypsindeficiency.
 6. The method according to claim 4, wherein the renaldisease is selected from the group consisting of diabetic nephropathy,focal segmental glomerulosclerosis (FSGS), hypertensive nephrosclerosis,chronic glomerulonephritis, chronic transplant glomerulopathy, chronicinterstitial nephritis, and polycystic kidney disease.
 7. The methodaccording to claim 4, wherein the cardiovascular disease is selectedfrom the group consisting of atherosclerosis, arteriosclerosis,dyslipidemia, hypercholesterolemia, and hypertriglyceridemia.
 8. Themethod according to claim 4, wherein the metabolic disease is selectedfrom the group consisting of insulin resistance, Type I and Type IIdiabetes, and obesity.
 9. A pharmaceutical composition comprising acompound of claim 1 and a pharmaceutically acceptable carrier.
 10. Acompound selected from the table below: Compound 2

4

5

or a pharmaceutically acceptable salt thereof.